(M1230-10-58) Evaluation of Niclosamide as a Repurposed Adjuvant for Treatment of Extensively Drug-Resistant Burkholderia cepacia Complex Infections

Purpose: Burkholderia cepacia complex (Bcc) is a group of closely related gram-negative bacteria that can cause severe opportunistic respiratory infections in patients with cystic fibrosis (CF). There are limited treatment options for Bcc lung infections due to Bcc’s de novo resistance to many classes of antibiotics, including polymyxins, and the ability of the bacteria to adapt to stressors in the environment and acquire resistance1,2, e.g., through expression of efflux pumps3. Failure to treat Bcc lung infections can result in a severe decline in lung function and dissemination of the infection and may preclude CF patients from undergoing lung transplants. Development of novel antibiotics for Bcc is costly, time-consuming, and risky. A promising alternative approach is the identification of existing FDA-approved compounds that exhibit either stand-alone antimicrobial activity or can enhance the activity of other drugs. Niclosamide is an off-patent drug that is FDA approved for the treatment of intestinal tapeworms. Though niclosamide is reported to not exhibit activity alone against gram-negative bacteria, it has previously been observed to exhibit synergistic activity in combination with colistin against several gram-negative bacteria, presumably due to colistin’s ability to disrupt efflux and its permeabilizing properties favoring intracellular accumulation of niclosamide4. However, the poor oral bioavailability and dose-limiting GI toxicity5 of the existing chewable oral formulation of niclosamide has limited its clinical utilization for respiratory infections. This problem could be potentially overcome through a targeted, inhaled drug delivery approach. The purpose of our study was to investigate the pharmacologic activity of niclosamide against Bcc alone and in combination with colistin and to evaluate the efficacy of an inhaled niclosamide formulation in the treatment of extracellular and intracellular Bcc infection. We hypothesized that niclosamide and colistin would exhibit synergistic activity against Bcc, and that this effect could be further potentiated through direct delivery of niclosamide to the airways.

Methods: Clinical strains of Burkholderia cenocepacia (A, B) and Burkholderia multivalorans (A, B) were obtained from the Michigan Medicine Division of Pediatric Pulmonology and were isolated from CF patient sputum. Occurrence of synergistic, additive, or antagonistic effects between niclosamide and colistin sulfate against the clinical strains was determined using a microdilution checkerboard assay as specified by CLSI using uncoated polystyrene microplates6. Mean inhibitory concentrations (MICs) were determined using a resazurin assay. As a positive control, the MIC of colistin was determined using a susceptible P. aeruginosa strain (ATCC Boston 41501). To evaluate the efficacy of niclosamide against Bcc within the context of the host cell environment, THP-1 monocytes were differentiated to macrophages, infected with B. cenocepacia isolate A, and treated with varying doses of a previously developed respirable formulation of niclosamide embedded in a carrier matrix consisting of human lysozyme, sucrose, histidine, and polysorbate 80 that was generated using a previously described method7. The aerosol performance of the inhaled niclosamide formulation was evaluated using a Next Generation Impactor and a medium-resistance RS01 dry powder inhaler (Plastiape) according to methods prescribed by USP <601 >8> and geometric size distribution of the formulation was evaluated using laser diffraction.

Results: Three out of the four Bcc strains tested were inhibited by niclosamide alone, while all four strains exhibited a decrease in MIC for both niclosamide and colistin when administered in combination (Fig 1). Though future investigations are required, this enhancement of niclosamide activity in combination with colistin may be due to the membrane permeabilization effects of colistin9. Susceptibility to niclosamide was not universal across the Bcc clinical strains tested, which may be due to mutations in efflux pumps or other differences in resistance mechanisms among clinical strains. Niclosamide, in combination with a respirable carrier formulation consisting of lysozyme and stabilizing excipients, also exhibited statistically significant intracellular antimicrobial activity at the highest dose tested (3.2 µg/mL). Compared to untreated controls, a 12 % decrease in bacterial number in cell lysate (p=0.0067) and a non-significant 6.5% decrease in cell culture supernatant (p=0.2683; representing extracellular efficacy) was observed after 24 hours of exposure to niclosamide particles at this concentration (Fig 2). The prepared niclosamide-lysozyme formulation was unimodal, which indicated that the niclosamide microparticles were fully incorporated into the carrier matrix (Fig 3). The emitted fraction (EF) of the formulation was 78%, respirable fraction (RF) < 5 µm was 58%, and RF < 3 µm was 45.7% using the RS01 medium resistance device at a 4 kPa pressure drop.

Conclusion: Niclosamide exhibits stand-alone antimicrobial activity and additive activity with colistin sulfate against several clinical strains of Bcc. Spray dried niclosamide-lysozyme particles exhibit efficient aerosolization using a simple dry powder inhaler and efficacy against intracellular Bcc infection. Future studies will focus on dose and formulation optimization to maximize antimicrobial activity within the host cell environment.

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8. United States Pharmacopeia (2022). General Chapter, 〈601〉 Inhalation and Nasal Drug Products: Aerosols, Sprays, and Powders—Performance Quality Tests. USP-NF. Rockville, MD: United States Pharmacopeia.
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Acknowledgments: We thank Dr. Lindsay Caverly for providing the four Bcc isolates from CF patients’ sputum.